Electrically polarizable biomaterials, structured, single-component polyelectrolyte layers, multicomponent polyelectrolyte multilayers or layers of preformed polyelectrolyte complexes, atoms, ions and/or molecules (epAIMP) are oriented in an electric field. A torque, which directs the dipole in the direction of the electric field, acts on the electric dipole of the epAIMP in a homogeneous electric field. In the inhomogeneous electric field, the dipole of the epAIMP is subject to a force, which pulls it into areas of higher field intensity.
The dynamics of the acting forces determines whether the forces of inertia, which counteract the motion of epAIMP in a homogeneous or inhomogeneous electric field, can be overcome. A recommended solution is the stick and slip method, i.e., the action of a slowly moving force in the desired direction and fast switch-off of the moving force.
A biomaterial is defined as a substance of natural or artificial origin that treats, improves or replaces any tissue, organ or any function of the body temporarily or permanently independently or as part of a whole (D. F. Williams: Definitions in Biomaterials. Progress in Biomedical Engineering, 4th ed. Elsevier, Amsterdam 1987).
Biomolecules are molecules of organic substances, which occur in living organisms. Biomolecules interact with biomaterials and define the biological environment for biomaterials. Biomolecules are defined in the sense of the present invention as all natural and artificial materials                that occur in living organisms or are formed by living organisms,        that are manufactured artificially and simulate natural, organic substances, or        to which other (artificial) substances or metals can be added or have been added and/or which have formed connections with other artificial or natural materials.        
Biomolecules may carry electrostatic charges and form an electric dipole. Such biomolecules are included among the epAIMP. The dipole-dipole interactions of the polar biomolecules ensure the orientation of the biomolecules among one another. On the nanometer length scale, the electrostatic interaction belongs to the forces with the greatest strength and also to those with the greatest range. Electrostatic forces are also very strong in aqueous solutions and thus keep the biomolecules away from each other.
In the study of biomaterials or biomolecules, the carrier must take up the biomaterials in a manner limitable in space and time and make the biomaterials available for local requirements. The adhesion to cells (adhesiveness to cells) of biomaterials and biomolecules on the carrier as well as cell mobility play an important role here. The underlying mechanisms of adhesion to cells or cell mobility have not been fully researched.
Distinction is made between biodegradable and non-biodegradable carriers for biomaterials.
Biomolecules may be hydrophobic or hydrophilic. Hydrophobic biomolecules are nonpolar. Hydrophilic biomolecules are polar and can be charged either positively (basic) or negatively (acidic).
The inverted optical or confocal microscope makes it possible to quantify the individual cell-cell and cell-surface interactions under physiological conditions. A plurality of important parameters of cellular adhesion, for example, maximum cell adhesion force, individual unfolding events, the tether characteristic as well as the total energy of the electrostatic bond can thus be determined.
To make it possible to carry out investigations on individual molecules by means of fluorescence detection on biomolecules under nearly physiological conditions, the biomolecules must have high fluorescence activity. Since very few molecules are intrinsically fluorescent or their fluorescence properties do not meet the requirements, biologically functional molecules are marked, as a rule, via covalent binding of stains developed specifically for this purpose (Sauer, Han et al., 1993).
Biomolecules with odd electrons have a magnetic moment that is stronger than the moment of a proton by about three orders of magnitude. This moment can be used as a probe in highly sensitive measurements with methods of electron spin resonance spectroscopy (EPR) in order to obtain information on a scale ranging from the atomic to the nanometer scale.
To construct biosensors, the biologically active elements must be fixed on carriers. Various methods are available for fixing, and distinction is made between physical and chemical methods. The physical methods include primarily adsorption. It is the simplest method. However, biosensors prepared in this manner respond sensitively to changes in the ambient conditions because of the reversible nature of the binding equilibrium. The chemical methods include covalent coupling and crosslinking Only the groups of the biomolecules that are not responsible for the biological activity may be involved in the covalent coupling with derivatized, water-insoluble carriers.
WO/2005/007387 A describes the nanomanipulation of the piezoelectric carrier by applying an electric voltage, which leads to a change in the contact area defined between carrier and biomaterial or biomolecule. The drawback is that these piezoelectric carriers must be thin so that the electric voltages applied do not become too high.
Despite the enormous technological advances made in the past years, there are only a small number of biosensory approaches with which a pharma screening can be carried out. The reasons for this are that the “classic” biosensor on the basis of enzymes and antibodies yields mostly only structural rather than functional information and the biomolecular interaction analysis with biosensors is very demanding. However, pharma screening can be given fresh impetus by microtechnologies and nanotechnologies by means of biosensors. In particular, the “High Content Screening” (HCS), a simultaneous screening of many effects, with biosensors appears to be feasible and could represent a way out of the currently developing bottlenecks in the development pipelines of many pharmaceutical companies.
Biosensors were developed in the past years for many different analytical tasks. Applications in the clinical medical area, fermentation control, quality control of foods and environmental analyses were in the foreground. However, the possibilities of using biosensory techniques are far more varied. On the one hand, highly specific structural elements, which are characteristic of potential drugs, can be recognized with biosensors; on the other hand, it is possible to carry out assays with which drug effects can then be detected. A biosensor typically comprises a biological recognition element and a physical sensor (transducer). Biosensors are usually classified according to the principle of function of their signal converter. Distinction is made essentially between electrochemical, optical, calorimetric and microgravimetric biosensors. However, such a classification is not meaningful in case of complex systems that contain whole living cells.
Polyelectrolyte materials have local electric dipoles, i.e., optionally positively or negatively charged functional groups, and can be bound with the forces or charge centers of the carriers acting as oppositely charged forces or charge centers by attractive electrostatic interaction (physisorption, adsorption). Such polyelectrolyte materials are included among the epAIMP.
Polyelectrolyte materials (PEL) [M. Schmidt (Vol. Ed.): Polyelectrolytes with Defined Molecular Architecture I, Springer, 2004] comprise single-component polyelectrolyte systems (PEE), whose charges have only one sign or which contain only one type of polyelectrolyte (e.g., negatively charged polyacrylic acid), as well as mixed polyelectrolyte systems, e.g., polyelectrolyte multilayers (PEM) or preformed polyelectrolyte complex particles (PEC). The different polyelectrolyte materials are shown schematically in FIG. 1.
Single-component PEL systems (PEE) are formed by simple adsorption from solutions of the respective PEL at a suitable concentration on the carriers. Mostly only inhomogeneous PEL layers (islands) are formed now in case of conventional substrates (lower surface charge density than those described here) because of the electrostatic self-repulsion between surface regions with already adsorbed PEL layers and the rest of the PEL in solution. The carriers in question here possess substantially higher surface charge densities (for values, see below). As a result, single-component PEL layers with substantially higher degree of occupation exceeding that in a monolayer can be formed or lead to adsorbed quantities of PEL that differ markedly from those that have hitherto been measured experimentally on highly charged substrates. As a result, new data can possibly be gained on the theory of the adsorption of polyelectrolytes on solid surfaces [e.g., Fleer et al., Polymers at Interfaces, Chapman, 1993, R. R. Netz, J. F. Joanny, Macromolecules, 32, 9013 (1999), A. V. Dobrynin, M. Rubinstein, Prog. Polym. Sci., 30, 1049-1118 (2005)] or these can be better harmonized with experimental findings on the dependence of the adsorbed quantity of PEL on the surface charge density.
By contrast, PEM are prepared by consecutive adsorption of polycations (PEL carrying positively charged functional groups or monomer units) with polyanions (PEL carrying negatively charged groups or monomer units) on a substrate, where the carrier is, for example, a silicon carrier, whose surface is treated chemically and/or physically [WO 2010 066432 A]. The cause of the adsorption and desorption is not isolated chemically and physically from the environment.
Polyelectrolyte complex particles (PEC) are formed at first by mixing a polycation and polyanion solution at a nonstoichiometric ratio in the volume phase [US 2008/0058229 A1]. The particles with neutral core and positively or negatively charged shell, which are formed in the process depending on the mixing ratio, can be bound to the charged carrier by physisorption similarly to the PEL.
PEM films can form oriented nanostructures by the use of rigid-chain PEL on, e.g., unidirectionally texturized carriers [M. Müller, Orientation of -helical Poly(L-lysine) in Consecutively Adsorbed Polyelectrolyte Multilayers on Texturized Silicon Substrates, Biomacromolecules, 2 (1), 262-269 (2001)].
Likewise, rod-shaped PEC particles, which can also produce oriented nanostructures on unidirectionally texturized carriers, can be formed by using rigid-chain PEL [M. Müller, T. Reihs, W. Quyang, Needle like and spherical polyelectrolyte complex nanoparticles of poly(L-lysine) and copolymers of maleic acid, Langmuir, 21 (1), 465-469 (2005)].
Oriented PEM films or PEC films can affect the cell growth and lead to the replacement of plasma-modified substrates, which can be prepared with a substantially greater effort.
Conductive PEL and polymers, e.g., polyaniline, polypyrrole, polythiophene or polyethylenedioxythiophene (PEDOT) can also be included and incorporated as single-component PEL materials or in PEM or PEC and used as conductive bonding agents between indium tin oxide (ITO) and active dye layers for organic light-emitting diodes (OLEDs).
These polyelectrolyte materials can, in turn, be used as bonding material for other materials, e.g., biomolecules and biomaterials, based on their structural relationship or even identity to biomaterials (proteins, polysaccharides, polynucleotides). Passivating layers that are inert in a controlled manner (PEL-1) or actively binding layers (PEL-2) or even biocidal layers (e.g., bacteria) (PEL-3) for biomaterials, biofluids or cells can thus form.
A question of current interest is the determination of the rate, strength and/or specificity of the binding, as well as the determination of the concentration of active biomolecules and particles as well as the identification of new interaction partners (“ligand fishing”) on polyelectrolyte materials. Biomolecules may be hydrophobic or hydrophilic. Hydrophobic biomolecules are nonpolar. Hydrophilic biomolecules are polar and may have either positively charged (basic), negatively charged (acidic) charge centers (functional groups in contact with the solvent or be electrostatically neutral. Some low-molecular-weight biomolecules such as amino acids, monosaccharides and nucleotides are reactive monomers for the polymerization into high-molecular-weight biomolecules, the biopolymers such as proteins (e.g., collagen, serum albumin, insulin), polysaccharides (e.g., glycogen, starch, cellulose, dextrans, chitin) and polynucleotides (e.g., DNA, RNA).
FIG. 1 shows different, already electrically polarized or electrically polarizable biomaterials (BM), atoms, ions and/or molecules before (MA, MB, MC) and after the modification (MA′, MB′, MC′), single-component polyelectrolyte systems (PEE), polyelectrolyte multilayers (PEM) and polyelectrolyte complex articles (PEC). No distinction is made in the figures between polarizable biomaterials (BM), atoms, ions or molecules and single-component polyelectrolyte systems (PEE), polyelectrolyte multilayers (PEM) and polyelectrolyte complex particles (PEC). The lower part of FIG. 1 shows the structure of the polyelectrolyte materials (PEL) with a positive excess charge, and the upper part shows the structure of the PEL with a negative excess charge. These are shown in the further figures by an oval with a “+” or “−” corresponding to their excess charge. These electrically polarizable particles are designated by epAIMP. The epAIMP, which are shown schematically in different sizes and shapes, differ in terms of their electric polarizability, their mass and their tendency to bind with other biomaterials, atoms, ions and/or molecules or polyelectrolyte materials. The carrier according to the present invention utilizes the electric polarizability of the epAIMP, which is an indicator of the shiftability of positive charge relative to negative charge. Electron clouds can now be shifted relative to the positive heavy nucleus in atoms and ions and/or positive ions relative to negative ions in molecules. More complex epAIMP with higher electric multipoles may also appear and interact electrostatically with one another. Only electric dipoles are shown in the drawings for simplification.
The epAIMP may have a permanent electric dipole and/or form an electric dipole in electric fields. The dipole-dipole interactions of epAIMP ensure the orientation of the latter relative to one another. The electrostatic interaction belongs to the interactions with the strongest force and with the greatest range on the nanometer length scale. Electrostatic forces are also very strong in aqueous solutions and thus keep epAIMP away from each other. The epAIMP may occur not only as a dipole but also as a more complex epAIMP with higher electric multipoles and interact with one another electrostatically. Only electric dipoles are shown in the drawings for simplification.
Motion of the epAIMP is possible in electric fields between two electrodes, to which an electric voltage is applied from the outside.
Individual mobile ions can be moved in a solid with rear-side electrode and with static or positionable front-side electrode, e.g., the metallically conductive measuring tip of an Atomic Force Microscope [N. Balke et al.: Nanoscale mapping of ion diffusion in a lithium-ion battery cathode. NATURE NANOTECHNOLOGY 5 (2010), 749-754].
Outside a solid, epAIMP can be moved by dielectrophoresis [Pei Yu Chiou et al.: Massively parallel manipulation of single cells and microparticles using optical images. NATURE 436 (2005) 370-372] between the surface of a carrier and a large-area front-side electrode, which is arranged at a spaced location from the surface of the carrier and does not touch this. The rear side of the carrier is illuminated for this through a shadow mask. The photogenerated charge carriers in the carrier locally increase the conductivity of the carrier and locally shift the position of the rear-side electrode from the lower ITO layer into the undoped a-Si:H of the carrier. The distance between the upper ITO layer (front-side electrode) with unchanged position and the rear-side electrode with variable position is locally changed hereby. The voltage dropping between the front-side electrode and the rear-side electrode causes a locally varying electric field. The direction of the electric field is determined by the polarity of the voltage applied to the front-side electrode and the rear-side electrode. Different positions of the rear-side electrode can be set on the micrometer scale very rapidly by changing the shadow mask.
A molecular machine is constructed from a discrete number of epAIMP and assumes special functions on the nanometer and/or micrometer length scale. Its mode of operation is similar in many respects to that of the machines created by humans. It is a mechanism in which individual parts fit into one another, move and interact in order to perform a certain task [Goodsell, David S.: Wirtschaft and Produktion in der molecularen Welt [Economy and Production in the Molecular World], Translated by Hummel, Isolde, 2nd ed., 2010. Spektrum Akademischer Verlag].
Molecular machines produce a mechanical motion (output) as a response to a specific stimulus (input). There are artificial and biological molecular machines.
All molecules that transmit functions from the meter length scale to the nanometer and/or micrometer length scale and perform same are called molecular machines in the broadened sense of the word.
The epAIMP for building the molecular machines exists in a small number of forms and sizes only. For example, artificial molecular machines can be built up from rotaxanes and catenanes Biological molecular machines are built up largely from six types of atoms, namely, carbon, oxygen, nitrogen, sulfur, phosphorus and hydrogen, as well as largely from four types of molecules, namely, proteins, nucleic acids, lipids and polysaccharides.
When atoms, ions and/or molecules meet each other, they interact with one another. If this interaction is weak, e.g., in slow neutral gases, the atoms and/or molecules continue their paths unchanged after the collision. If the interaction is strong, e.g., in gases containing ions or in fast neutral gases, the atoms, ions and/or molecules continue their paths in a changed form after the collision, during which coulomb forces may act between ions and resonant charge transfer may act between neutral atoms and molecules.
The discrete number of atomic, ionic and/or molecular components is composed into artificial and biological molecular machines, which are always tailored optimally to a certain role, according to a blueprint by means of the basic concepts, namely, chemical complementarity and hydrophobicity.
If the interaction is complementary, e.g., if atomic and molecular areas fit perfectly into a similarly shaped atomic and molecular area of an adjacent molecule, a solid bond is formed.
Molecular machines use two special types of bonds: Hydrogen bridges between a hydrogen atom and an oxygen or nitrogen atom, as well as salt bridges between atoms, ions, and/or molecules, which carry an opposite electric charge. These special bonds function like small clamps, which couple atoms, ions and molecules with one another.
The formation of a special type of bond can be catalyzed by a modification of the epAIMP.
A large-area modification of heat-sensitive epAIMP can be brought about by supplying heat. The heat energy can be produced, e.g., by reversing the magnetic poles of magnetizable particles in a magnetic field applied from the outside [US 2008 0319247 A1], by absorption of electromagnetic waves and/or by excitation of lattice vibrations by laser pulses of a pulse length of 10 nanosec with an energy density of 20 W/mm2 [M. E. Msall et al.: Ballistic phonon production in photoexcited Ge, GaAs, and Si. PHYS. REV. B. 65 (2002), 195205-1-7].
The simple artificial molecular machines already synthesized include motors [Javier Vicario et al.: Controlling the speed of rotation in molecular motors. Dramatic acceleration of the rotary motion by structural modification, CHEM. COMMUN. (2005), 5910-5912], propellers [B. Wang et al.: Chemically Tunable Nanoscale Propellers of Liquids. PHYS. REV. LETT., 98 (2007), 266102-1-4], switches [Jean-Pierre Desvergne et al.: Cation complexing photochromic materials involving bisanthracenes linked by a polyether chain. Preparation of a crown-ether by photocycloisomerization; J. CHEM. SOC., CHEM. COMMUN. (1978), 403-404], molecular transporters, molecular pincers, molecular sensors and logic elements [Jonathan E. Green et al.: A 160-kilobit molecular electronic memory patterned at 1011 bits per square centimeter. NATURE, 445 (2007), 414-417].
The manufacture of molecular machines is an essential prerequisite for current research in the field of molecular assembling, DNA machines, nanoelectromechanical systems, nanosensors and protein dynamics.
Complex molecular machines were already designed theoretically, but could not be tested as yet, because there are no methods so far for manufacturing complex molecular machines.
Molecular sensors interact with an analyte and indicate a measurable change. Fluorescences in ether radicals on molecular sensors are frequently used to measure the change after interaction.
The carriers developed so far are not suitable for taking up, modifying and moving individual epAIMP in a manner limited in space and time. No molecular machines can be manufactured with the hitherto known carriers from the electrically polarizable atoms, ions, molecules, biomaterials or polyelectrolyte materials.
The direction of the electric field at a given point in time is the same everywhere during electrophoresis [Pei Yu Chiou et al.: Massively parallel manipulation of single cells and microparticles using optical images. NATURE, 436 (2005), 370-372]. The photogenerated charge carriers also diffuse into the undoped a-Si:H in case of excessively long illumination of the rear-side electrode, they increase the conductivity of the undoped a-Si:H everywhere and thus shift the position of the rear-side electrode over a large area from the lower ITO layer into the undoped a-Si:H. In addition, the application of an electric voltage between the front-side electrode, which is located at a spaced location from the surface of the carrier (nitride layer), and the rear-side electrode, is disadvantageous, because the distance between the nitride layer and the front-side electrode must be greater than the particles or cells in order for the latter to be able to move mechanically freely on the nitride layer.
The propagation of the electric field between the front-side electrode and the rear-side electrode is not homogeneous during electrophoresis, because the particles and/or cells pass on the electric field lines differently due to their dielectric properties than does the surrounding material. The particles and/or cells thus affect the electric field, which shall actually be used to affect the particles and/or cells.
Smaller particles and/or cells (<1 micrometer) cannot be sorted during electrophoresis, because the localization of the rear-side electrode is limited due to the diffusion of the photogenerated charge carriers and because the electric field between the front-side electrode and the rear-side electrode is not large enough to overcome the Brownian motion of the particles and/or cells.
The detection of the localized particles and/or cells can be carried out during electrophoresis only optically through the front-side electrode. If the front-side electrode is taken away, no electric field will develop and the particles and cells cannot be localized. Detection of the localized particles by means of atomic force microscopic measurements or by means of scanning electron microscopic measurements is not possible through the front-side electrode.